Protective Effects And Mechanisms Of Etidronate-zinc Complex In 2-vessel Occlusion Model Rats

BackgroundChronic cerebral hypoperfusion(CCH)is a major cause of cognitive-related diseases and promotes the progression of neurodegenerative diseases.Two-vessel occlusion(2-VO)has been widely used to mimic CCH in rodents.In addition,neuroinflammation has an important influence on cognitive impairment caused by 2-VO.Studies have found that inhibition of neuroinflammation may have a better therapeutic effect on cognitive impairment caused by 2-VO.Bisphosphonates are a class of drugs that target bone disease-related diseases such as Paget's disease and osteoporosis.Etidronate-zinc complex(Eti-Zn)is a new hybrid.At present,the effect of Eti-Zn drug on CCH has not been reported.In this study,we investigated the effects of Eti-Zn on learning,memory,and synaptic plasticity in 2-VO model rats.We further explored the underlying mechanisms for investigating whether this effect is associated with neuroinflammation.MethodsThere were three groups: Sham group,2-VO group and 2-VO+Eti-Zn group.The CCH was induced by 2-VO in adult male Sprague-Dawley rats.Eti-Zn(20 mg/kg/day,i.v.)was administered for 7 days after two-week operation.The same amount of normal saline was injected into other rats.After treatment,a series of tests were carried out.The rats of different groups were trained in Morris water maze to evaluate the spatial learning and memory abilities.Experimental results also could demonstrate the flexible memory in rats.Novel object recognition was performed to assess the animal's ability to distinguish objects.LTP and DEP were recorded from Schaffer collaterals to CA1 of hippocampus to measure the changes of synaptic plasticity.In addition,Golgi-Cox staining is an important means of observing dendritic spine density.We also detected the expressions of synapse-associated proteins in the hippocampus.The levels of neuroinflammatory in hippocampal tissues of three groups' rats were measured.Western blot analysis revealed that phosphorylated NF-?Bp65 level.We detected the expression of Iba 1 and nuclear translocation of NF-?Bp65 by Immunofluorescence staining.Results(1)The Morris water maze test indicated that the escape latencies were all decreased during the 4 days of the IT in the three groups.Injection of Eti-Zn significantly shortened the treated rats' escape latency,increased numbers of platform crossings and the percentage of time spent on the target quadrant.However,there was no significant difference in swimming velocity between these three groups.We investigated the spatial cognitive impairment that underwent bilateral ligation of the carotid arteries.Eti-Zn administration ameliorated the cognitive impairments induced by 2-VO.(2)With Eti-Zn treatment,the escape latencies in the RT stage were rescued significantly.The Eti-Zn significantly increased the number of platform crossings and the target quadrant residence time of the treated 2-VO rats in the RET phase.The test suggested that Eti-Zn effectively alleviated the reversal of spatial memory in 2-VO model rats.(3)In the NOR test,a lower recognition index was observed in 2-VO rats compared with the Sham group in long-memory testing phase.Furthermore,the recognition index of new objects was increased significantly in 2-VO rats treated with Eti-Zn.Our study suggested that Eti-Zn effectively alleviated recognition memory impairments of 2-VO model rats.(4)We examined the LTP and DEP from the Schaffer collaterals to the CA1 region.The result demonstrated that the fEPSP slopes of LTP were reduced after 2-VO,however,Eti-Zn reversed the degradation.The fEPSP slopes of DEP in 2-VO group were significantly larger than that of the Sham,while the fEPSP slopes were evidently reduced after treatment with Eti-Zn.(5)Furthermore,Eti-Zn significantly upregulated NMDA receptor(NR)2A,NR2 B,postsynaptic density protein 95 and synaptophysin levels and prevented the destruction of the dendritic spine.(6)Moreover,Eti-Zn treatment declined the overactive microglia and reduced the neuroinflammatory cytokines in hippocampus.(7)In CCH,nuclear translocation was sharply enriched in NF-?Bp65.Phosphorylated NF-?Bp65 levels were up-regulated.Eti-Zn reduced nuclear translocation,and COX-2/iNOS proteins regulated by NF-?B dramatically.Those results further confirmed that Eti-Zn could suppress neuroinflammatory responses by inhibiting the NF-?B pathway.Conclusions(1)These results demonstrated that the administration of Eti-Zn decreased ischemia-induced nervous system injury and improved postoperative learning and memory abilities.(2)Our results suggest that Eti-Zn may reverse the synaptic plasticity and cognitive impairments by reducing the neuroinflammation in 2-VO model rats.